A majority of cancer-related deaths result from metastasis, or the spread of tumor cells throughout the body. One of the initial steps in establishing a metastatic potential in tumor cells is the epithelial to mesenchymal transition (EMT), during which epithelial cells undergo transcriptional reprogramming that results in the acquisition of mesenchymal characteristics, such as motility, and an invasive phenotype. Determining the mechanisms that coordinate the switch in transcription profiles during EMT is necessary to identify new targets and develop therapies that effectively inhibit metastasis. Epigenetic alterations are important factors contributing to the aberrant transcription potential that drives metastatic processes, such as EMT. Additionally, RNA Polymerase (Pol) II promoter-proximal pausing is a key regulatory mechanism for transcription that refines expression levels and poises genes for signal-induced transcriptional changes. Our laboratory has recently identified a novel epigenetic mechanism regulating Pol II pausing. Trimethylation of histone H4 lysine 20 (H4K20me3) by the methyltransferase SUV420H2 induces Pol II pausing by preventing histone H4 lysine 16 acetylation at a select set of genes. This local gain in H4K20me3 is seen in mesenchymal breast cancer cells and not epithelial breast cancer cells. In addition, downregulation of SUV420H2 and decreased H4K20me3 confer a poor prognosis in multiple types of cancer and is associated with the mesenchymal phenotype in primary breast cancer. Furthermore, in epithelial ovarian cancer cells, H4K20me3 marks several normally repressed genes, and depletion of H4K20me3 leads to their re-activation, supporting a role for histone H4 modifications in EMT reprogramming. However, genome-wide changes in SUV420H2 and H4K20me3 during EMT remain uncharacterized. Moreover, whether SUV420H2 targeting is sufficient to drive local changes in transcription is unclear. In this proposal, we will use genome-wide and locus specific techniques to test our hypothesis that the transcriptional reprogramming that drives EMT involves the redistribution of SUV420H2 and H4K20me3, which alters the transcription potential of cancer cells. The specific aims of the proposed research are 1) to determine the impact of SUV420H2 downregulation on EMT, 2) to determine the genome-wide redistribution of SUV420H2 and H4K20me3 and its impact on Pol II pausing and transcription during EMT, and 3) to determine whether SUV420H2-targeting and H4K20me3-enforced Pol II pausing are sufficient to induce gene repression at targeted loci in epithelial cancer cells. The overall goal of these studies is to determine the role of SUV420H2 and H4K20me3 redistribution in the transcriptional reprogramming that occurs during EMT. In the long-term, our proposed studies will provide insight into the epigenetic mechanisms that govern aberrant transcription in cancer and allow for the development of novel therapies and drug combinations to target metastatic progression.